The interest in the use of monitored fecal steroids as an indicator of the endocrine status has grown considerably in recent years for an ample variety of species. Nevertheless, the success in the application of the basic technique requires the knowledge of the time at which the animal already presents hormonal secretion, as well as the metabolic forms in which these steroids are excreted (time lag).
The time lags are necessary to determine the time between the occurrence of physiological events and its detection in the feces. The knowledge of the metabolic events is critical to maximize the efficiency of the immunoassays used to quantify the feces steroid profiles.
Wasser, S. K; Velloso, A. D. L; Rodden, M. D. 1995. Using fecal steroids to evaluate reproductive function in female maned wolves. Journal of Wildlife Management, v. 59, n. 4:889-894; they made a study that represented the first attempt to apply the measurement of canine feces steroid and suggested that these noninvasive measurements can provide an important tool for the herd wolf pack in danger of extinction.
For this, they measured the estrogen and progestin concentrations during a period of time in the feces of nine female wolves to determine if the measurements can be used to observe their reproductive function. The subjects were located in five zoo parks of the United States of America, two to three samples were collected weekly for a period of 3 to 5 months, beginning just in the period previous to the heat stage.
The measurement of fecal steroid indicated occurrence of ovulation and pregnancy in 8 of the 9 females. The females in cycle showed estrogen presence with the production of progesterone in the luteum phase. This estrogen presence was followed by a maintained production of progestin levels of 27.2±2.65 mu-g/g in conceptive cycles during the next subsequent 63 days (first period of gestation), which was higher than the 7.6±0.73 mu-g/g of the progestin concentration in non-conceptive cycles.
The estrogen concentrations in acyclic young females never reach more than 50 ng/g (nanograms per gram), whereas their values of progestin remained under 3.0 ng/g. The measurements of fecal steroids provide useful means to characterize the fertility, including the ovulation and the pregnancy in female wolves.
Brown, J. L; Wasser, S. K; Wildt, D. E; Graham, L. H. 1994. Comparative aspects of steroid hormone metabolism and ovarian activity in fields, measured noninvasively in feces. Biology of Reproduction, v. 51, n. 4:776-786; used noninvasive fecal tests to study the steroid metabolism, as well as the ovarian activity of several feline species.
Using the domestic cat Felis catus as a model, when estradiol (E-2) (14C) and the progesterone (P-4) (14C) were injected, the excreted products were determined. It was found on a two day period activity of 97.0±0.6% and 96.7±0.5% of E-2 and P-4. E-2 was excreted as non-conjugated estradiol and as estrogen (40%) and also as non-hydrolizable conjugated enzyme (60%). In addition, P-4 was excreted initially as a non-hydrolizable enzyme, as well as non-conjugated epimers of pregnenolone.
In view of the repeated capture and the taking of blood samples, which are generally impractical strategies in the monitoring of the reproductive stratum of wild species, the non-invasive methods to pursue the reproductive activity have been increased significantly.
Wasser, S. K; Thomas, R; Nair, P. P; Guidry, C; Southers, J; Lucas, J; Wildt, D. E; Monfort, S. L. 1993. Effects of Dietary Fibre on Faecal Steroid Measurements in Baboons Papio-Cynocephalus-Cynicephalus. Journal of Reproduction and Fertility, V. 97, n. 2:569-574; used radioimmunoassay (RIA) of pregnanediol-3 alpha glucuronic (PdG) in the urine, conjugated of estrogens, fecal progesterone and estradiol to assure the menstrual cycles and the pregnancy of captive elks.
Using PdG in the urine, different reproductive cycles that began in October were identified when the estral behavior agreed with the maximum level of excretion of PdG. Although this compound increased more than five times over the cyclical levels of the pregnancy, the concentrations were variable making the pregnancy diagnosis incorrect when using this method.
The estrogen conjugated was not useful to monitor the heat cycles; nevertheless, during the last month of gestation, the conjugated estrogen levels in the urine increased less than five nanograms per milligram of Creatinine (Cr) to more than 50 ng per milligram of Cr, making this a useful method for the definitive detection of the delayed pregnancy.
In order to establish a simple pregnancy test, they evaluated estradiol and progesterone in feces (one to six samples per individual), collected over a period of two years, of 16 elks of different ages, kind and physiological classifications, pregnant against non-pregnant.
Using fecal progesterone and blind tests; the technicians correctly identified the status of pregnancy in 22 of the 26 cases (85%) with three false positives and a mistaken diagnosis; fecal estradiol proved to be less effective (15/26) (58%) for the exact diagnosis of pregnancy. These methods have excellent potential to monitor the reproductive activity of elks in captivity and wildlife.
Barney A. Schlinger and Arthur P. Arnold. 1992. Circulating estrogens in a male songbird originate in the brain. Department of Psychology and Laboratory of Neuroendocrinology of the Brain Research Institute. University of California. Los Angeles, Calif.; observed that the gonad steroids act in the brain to regulate the development and expression of the reproductive behavior of vertebrates.
In addition, the steroids controlled in the brain are an integral part of the regulation of the feedback of the steroidogenesis. The actions of androgens in the brain are frequently mediated for the enzymatic activation or inactivation of the circulating hormone, including local conversion of androgen to estrogens.
They reported that the brain synthesizes great amounts of estrogen from androgens and releases the estrogen in the blood, being probable that the brain controls the levels in the plasma of this steroid, when totally contributing with high levels of estrogen found in the circulation.
Barney A. Schlinger and Arthur P. Arnold. 1991. The brain the major site of estrogen synthesis in a male songbird. Department of Psychology and Laboratory of Neuroendocrinology of the Brain Research Institute. University of California. Los Angeles, Calif.; found that the neural system that controls the singing of the birds can experience strong morphologic and functional changes during its development and adult stage and many of these changes are regulated by estrogenic hormones.
High levels of estrogen circulate in the blood of the males in singing birds and is present after the castration. These investigators measured the activity of aromatase, the enzyme that converts androgens in estrogens in several female tissues and adult males and as they had expected, the activity of aromatase was present in the male hypothalamus/preoptic region, the pituitary and the feminine ovary, although the aromatase was unusually active in telencephalon of males and females. On the contrary, the activity was undetectable in adrenal testis or other masculine tissue.
These results suggest that the brain is the source of the circulating estrogens in males and that its action over the singing system results from the local aromatization more than peripheral.
Schlinger and Callard, G. 1987. A comparison of Aromatase, alfa, and 5 beta-Reductase Activities in the Brain and Pituitary of Male and Female Quail (C. c. japonica). The Journal of Experimental Zoology. 242: 171-180; concluded that in numerous species of vertebrates, including the Japanese quail, the actions of the testosterone (T) on the neuroendocrine tissue were mediated in part by the conversion to estrogenic and androgenic metabolic.
The objective was to investigate which processes were favored in each identified androgenic tissue in the brain of the quail and if sexual differences were detected.
These investigators designed a test to quantify simultaneously aromatase 5α and 5β-reductase. In addition, they made the first definitive identification of aromatase in the pituitary of the quail and compared the three enzymatic activities of the pituitary of males and females.
The enzymes were measured in homogenized tissues by the conversion of (3H) androstenedion to (3H) estrone, 5α-androtanedion, and 5β-androtanedion. The activity of aromatase was restricted to the limbic tissue (previous hypothalamus, posterior hypothalamus, septum archistratium), which previously contained dyed nucleus; whereas the hiperestriatum, the cerebellum and the middle brain contained interfollicular nucleus, which were negative to aromatase.
The quail pituitary aromatized androgens to rates equivalent to the posterior hypothalamus or preoptical area (aHPOA). The 5α and the 5β-reductase were present in all studied tissues. Aromatase was significantly higher in aHPOA and in addition it was present in the pituitary of males, whereas 5α-reductase was significantly higher in the pituitary of the females.
This data suggests that a metabolic androgen complex of enzymes controls the neuroanatomic distribution (space) of the active hormone in the neuroendocrine tissue, and that the quantitative differences between males and females can serve to differentiate the behavior from sexes.
For the complete expression of testosterone T in the brain and in the peripheral tissue, the circulating hormone is transformed in situ to a metabolite that is biologically more active, for which three metabolic processes in the brain have been identified.
The aromatization and reduction 5α lead to the synthesis of estradiol-1β and 5α-dihidrotestosterone respectively. Each one of these metabolites is united to a separated receptor system and has unique actions in the brain in the process of sexual differentiation, sexual behavior and secretion of the pituitary hormone. For example, in the adult of the Japanese quail (Coturnix coturriii japonica), the aromatization of androgens to estrogens seems to be essential in the male mating behavior, the aggressiveness and the locomotive activity. Whereas the testosterone, per se, and the androgens 5α stimulate the singing and swagger.
The third process of the 5α reductase is presumed because of the inactivation that leads the 5β-DHT production (dihidrotestosterone) which does not have a mechanism of demonstrated biological activity in the brain or the pituitary. Wada. M. 1984. Effects of ventricularly implanted sex steroids on calling and locomotor activity in castrated male Japanese quail. Horm. Behav. 18:130-139.
Benjamin et al. 2002. Method For Sex Determination of Mammalian offspring. U.S. Pat. No. 6,489,092 B1. In this a work that is close to the present invention, which is directed towards a method to increase the percentage of descendants of one or another sex in mammals, by the contact of a spermatozoid sample with a specific antibody corresponding to the sex that is wanted to select, the antibody is united to a magnetic sphere of a diameter that produces the separation of a spermatozoid that has the sufficient motility to allow the insemination and the fertilization in a successful way.
Another invention that is related is: Malecha et al. 2004 Methods of Isolating the Androgenic sex Hormone From Crustacean Prawn and Marine Shrimp and Methods of Use. U.S. Pat. No. 6,740,794 B1.
This is a technique for the secretion in vitro of androgenic hormone (AH) of androgenic gland (AG) of the fresh water male shrimp in defined culture medium. One found that AH can be used in the manipulation of the shrimp reproductive process for the production of individuals of a determined sex, that is to say monosexual progeny.